The polysaccharide capsule of Streptococcus pneumoniae is the major virulence determinant of this organism. Despite early studies of the genetics, pathogenesis, and immunology of capsular polysaccharides, it remains unclear why certain capsular types appear to have a greater capacity to cause disease. Of the more than 80 known capsular serotypes, 23 account for more than 90% of all pneumococcal infections. The basis for emergence of new capsule types remains equally obscure. Whether influenced by mutation, recombination, or immune selection, genetic exchange of DNA among S. pneumoniae strains is likely to have played a major role in the evolution of capsular types. The long range goals of the proposed studies are to determine the effect of capsular type on the virulence of Streptococcus pneumoniae, to determine the genetic organization and regulation of capsule genes, and to determine the basis for genetic switching of capsule type gene cassettes. These studies will allow for the development of more effective strategies for treating and preventing pneumococcal infections, as they reveal how the capsular type of an invading strain might influence the effectiveness of vaccines or other therapies. Genetic and now molecular studies have indicated that the capsule type- specific genes are linked in the chromosome and that they are transferred and exchanged as a unit during genetic transformation. The genetic locus responsible for type 3 capsule expression has been identified through mutation, cloning, and sequence analyses. Flanking, non-type-specific DNA, present in all strains and likely to be involved in the recombination and switching of capsule types, has been located at one end of the region. In the proposed studies, the type 3 locus will be further characterized with respect to the type-specific genes, the non-type- specific DNA flanking each side of the region, and the factors involved in regulation of type 3 capsule expression. Genetic exchange of the chromosomal regions containing the type-specific genes has allowed us to construct near isogenic strains differing in the capsule type expressed. Initial studies indicate that virulence may be altered by the capsule type but non-capsular factors influence how different serotypes affect virulence. In the proposed studies, isogenic strains representing different capsular and genetic backgrounds will be constructed using defined PCR- or restriction fragments that contain the type-specific gene cassettes. These strains will be used to determine the effect of capsule type on mouse virulence and on in vitro measures of pneumococcal virulence, including phagocytosis and complement fixation. These studies will ultimately allow us to characterize the capsule genes of all capsular types, and to identify the structural features resulting in differences in virulence.